Host defense mechanisms have evolved as barriers to retroviral infections. It is important to understand the mechanism of these innate restriction factors in order to develop antiviral therapies and to determine how to protect against zoonotic retroviral infection. Studies have identified TRIM5alpha which blocks retroviral infection by targeting the viral capsid after entry, but before completion of reverse transcription. The actual mechanism of how TRIM5alpha restricts infection is unknown. Recent studies have suggested that TRIM5alpha accelerates uncoating of the HIV-1 core and that the proteasome is involved. The goals of this study are two-fold: (1): To determine if TRIM5alpha mediates uncoating of the HIV-1 core; (2): To determine if TRIM5alpha-mediated restriction of HIV-1 core involves the ubiquitin proteasome pathway. Aim 1 will test disassembly of the HIV-1 core in vitro in the presence and absence of lysates of cells over-expressing TRIM5alpha. Aim 2 will test whether TRIM5alpha induces degradation of purified HIV-1 cores in vitro using a system containing active cell fractions of the ubiquitin proteasome system. Ubiquitylation of viral core proteins will also be tested in vitro using cellular extracts capable of supporting Ubiquitylation of exogenous substrates. The extent of Ubiquitylation and degradation of viral proteins will be detected by immunoblotting and p24 ELISA. By establishing relevant assays for TRIM5alpha function in vitro, the proposed studies will open new avenues for further mechanistic analysis of TRIM5alpha restriction of retroviruses. Identification of the molecular mechanism of HIV-1 restriction by TRIM5alpha may reveal novel targets to further inhibit HIV-1 infection by preventing integration of HIV-1 DNA into the host cell chromosome. [unreadable] [unreadable] [unreadable]